 FRUCTOSE - metabolize for the energy

CHAPTER 11: needed for the flagella to propel them

through the female reproductive tract.

SEMEN - major nutrient of spermatozoa, so in the

absence of fructose sperm do not display
motility in thesemen analysis
ANALYSIS  FLAVIN- responsible of the gray
appearance of the semen
REASONS WHY WE NEED TO
EXAMINE SEMINAL FLUID? o Prostate gland

 To investigate the causes of infertility in  Aids in propelling the sperm trough the
marriages. urethra by contraction during ejaculation,
approximately 20-30 percent of the semen
 To check the effectiveness of previous volume is acidic produce in the prostate
vasectomy. gland
 For medico legal cases.  PG provides enzymes and proteins for
FORMATION AND PHYSIOLOGY coagulation and liquefaction

o Seminiferous tubules of testes ENZYMES

 site of spermatogenesis  Acid phosphatase - distinguishes semen

from other fluid
o Interstitial cells of Leydig of testes
 Zinc – decrease amount of these in
 produces and secrete testosterone seminal fluid has been associated with
disorders of the prostate gland
o Epididymis
 Potassium
 where sperm matures and develop flagella
 Citric acid
 sperm is being stored here (approximately
90 days) before ejaculation store  Ascorbic acid
approximately 90 days;
 Proteolytic enzymes - control the
 also produces fluid buy only 5- 10 % liquefaction and coagulation of seminal
fluid.
o Vas/ ductus deferens
LIQUEFACTION
 propels sperm to the ejaculatory duct
- Is the process when
o Seminal vesicles
the gel formed by proteins from the
 produce most of the fluid present in
semen (60- 70 %) this fluid is the transport seminal vesicles are broken up and the
mediumfor the sperm, it contains a high semen becomes more liquid
conc. Of fructose and flavin
 Stamin and Colin - they are from
prostate gland and an anti-bacterial  Times of specimen collection and receipt
o Bulbourethal glands METHODS OF COLLECTION
 Located below the prostate gland it add  Masturbation/ self- production
alkaline mucus to neutralize prostatic acid
 Coitus interruptus
and vaginal acidity
 Vaginal vault aspiration
 Produce 5% of the semen volume
 Condom method
 It is important for the semen to become
alkaline to neutralize the vaginal acidity SPECIMEN HANDLING
present as a result of normal bacterial
vaginal flora  All semen specimens are potential
reservoirs for HIV and hepatitis viruses.
SPECIMEN COLLECTION
 Standard precautions must be observed at
 First portion of the ejaculation is missing, all times during analysis.
then: SPERM COUNT WILL BE
DECREASE  Specimens are discarded as biohazardous
waster.
 Last portion of the ejaculation is missing,
then: SEMEN VOLUME IS DECREASE  Sterile materials and techniques must be
used when semen culture is to be performed
ABSTINENCE = 2days (no longer than 7 or when the specimen is to be processed for
days) bioassay, intra-uterine insemination, or in
vitro fertilization.
PRESERVATION
PROLONGED ABSTINENCE:
 Keep specimen at room temperature if
 Higher volume
needed to be transported
 Decreased sperm motility
 Specimen must be kept at body
 Increased flavin temperature while awaiting analysis
**who recommends that 2-3 samples be  For artificial insemination, it can be
collected not less than 7 days or more than 3 prevented in frozen state and stored for one
weeks apart, with two abnormal samples year at 85 degree Celcius
considered significant
 Specimens for fructose levels should be
RECORDS: tested within 2 hours or frozen to prevent
fructolysis.
 Patients name
NORMAL VALUSE FOR SEMINAL
 Birthdate
FLUID (MACROSCOPIC)
 Period of sexual abstinence
 COLOR: grayish white to pearly white
 Completeness of the sample and translucent

 Difficulties with collection  VOLUME: 2-5 ml per ejaculation

 ODOR: fishy, distinct, Clorox like and - Proteolytic enzymes such as alpha
musty odor chymotrypsin of bromelain
 VISCOSITY/ CONSISTENSCY: highly  pH
viscous Poursin droplets
- Increased: infection within the
 LIQUEFACTION TIME: 30-60 mins
reproductive tract
 Ph: 7.2 to 8.0
- Decreased: associated with increased
prostatic fluid, ejaculatory duct obstruction,
or poorly developed seminal vesicles
VARIATIONS:
NORMAL VALUSE FOR SEMINAL
 COLOR
FLUID (MICROSCOPIC)
- Rusty red to brown: presence of RBC
o SPERM MOTILITY: >50% within 1 h
- Yellowish: urine contamination, antibiotic,
o SPERM CONCENTRATION: >20
prolonged abstinence, pyrospermia
million/mL
 CLARITY
o SPERM COUNT: >40 million/ejaculate
- Clear: infertility
o MORPHOLOGY
- Turbid: infection and increased WBCs
 Kruger’s Strict Criteria: >14% normal
 VOLUME forms (strict criteria)

- Increased: prolonged abstinence  Routine Criteria: >30% normal forms

(routine criteria)
- Decreased: infertility, improper
functioning of semen- producing organs, o ROUND CELL: <1.0 million/mL
incomplete specimen collection
MICROSCOPIC EXAMINATION
 VISCOSITY
 Should be assed using a well-mixed,
- Rated as 0(watery) to 4(gel- like) or liquefied semen specimen within 1 hour of
specimen collection
- Reported as low, normal or high
 Performed in an undiluted specimen
 LIQUEFACTION TIME under ~20 HPF
- Incomplete liquefaction will impede  Evaluate by both speed and direction
sperm motility
SPERM MOTILITY GRADING
- Failure of liquefaction should be reported
Treatments for specimen that has not
liquefied after 2 hours: GRADE WHO criteria

- Equal volume of physiologic Dulbecco’s SPERM MOTILITY ACTION

phosphate buffered saline
- 4.0 a Rapid, straight-line motility
- 3.0 b Slower speed, some lateral SPERM CONCENTRATION AND SPERM
movement COUNT
- 2.0 b Slow forward progression, noticeable 2 TYPES OF COUNTING CHAMBERS:
lateral movement
 MARKER:
- 1.0 c No forward progression
 Undiluted specimen, sperms are
- 0 d No movement immobilized by heating, sperm motility is
assessed in the unheated portion
ALTERNATIVE SPERM MOTILITY
GRADING CRITERIA  NEUBAUER HEMOCYTOMETER
- Progressive motility (PM) Sperm moving  Counted in 4 corner squares and center
linearly or in a large circle square of the large center square
- Nonprogressive motility Sperm moving  Both sides of the hemocytometer are
with an (NP) absence of progression loaded and allowed to settle for 3 to 5
minutes; then they are counted, and the
- Immotility (IM) No movement
counts should agree within 10 percent
FACTORS AFFECTING MOTILITY:
 An average of the two counts is used in
 Complete liquefaction of seminal fluid the calculation
 Temperature  If the counts do not agree, both the
dilution and the counts are repeated
 Period of abstinence
 Manner of collection/ preparation
**counts are performed using brightfield/
 Number of cell present phase-contrast microscope.
 Type of movement of sperm cells  Round cells - are immature sperm and
 Chemical toxins WBCs

 Prescription drugs Peroxidase - positive granulocyte are the

predominant form of leukocyte in semen
FORMULA:
Computer Assisted Semen Analysis (CASA)
CNXS
 Provides objective determination of both
sperm velocity and trajectory (direction of 100
motion) N- number of spermatids of neutrophils
 Sperm concentration is also included in counted per 100 mature sperm
the analysis S- sperm concentration in millions per
 Found primarily in laboratories that milliliter
specialize in andrology and perform a high DILUTION:
volume of semen analysis
Diluting fluids:
 Sodium bicarbonate Using a 1:20 dilution an average of 60
sperm are counted in the five RBC counting
 1% formalin
squares on both sides of the hemocytometer.
 cold distilled water or tap water Calculate the sperm concentration per
milliliter and the total sperm count in a
CLINICAL SIGNIFICANCE: specimen with a volume of 4 mL. 60 sperm
 Azoospermia ccounted x 1,000,000 = 60,000,000 per/mL

 Complete or absence of spermatozoan in

the semen
 Necrospermia SPERM MORPHOLOGY
 Presence of increase number of sperm  Stain and evaluated at least 200 sperm
cells that are either completely dead or cells under oil immersion
immobile
 Evaluated with respect to both head and
 Oligospermia tail
 Deficiency in the number of sperm cells  Head
or presence of few motile cells
 Oval shaped head: ~ 4um long and 3 um
CALCULATING SPERM wide
CONCENTRATION AND SPERM
COUNT  Acrosomal cap: critical ovum penetration
since enzyme- containing located at the tip
SPERM CONCENTRATION: of the head
 Short cut method:  Middle piece
 Using 5 RBC squares  ~7.0 um long and is the thickest part of
# of sperm counted x 1 million = sperm in the tail
million/ mL  Tail
 Using 2 WBC squares  Flagellar, ~45 um long
# of sperm counted x 100, 000 = sperm in STAINS FOR SPERM MORPHOLOGY:
million/ mL
 Papaniculao’s
 Neubauer count chamber
 Giemsa
Sperm/uL = # of sperm counted x dilution #
of squares couned x vol. of square  Wright’s

*To convert sperm/uL to sperm/mL,  Shorr

multiply it by 1000
The head, neck, tail is measured using a
TOTAL SPERM COUNT micrometer.

 # of sperm/ mL x vol. of semen

>14 % normal morphology = Kruger’s strict  The presence of antisperm antibodies in
criteria female partner will result in a normal semen
analysis accompanied by continued fertility
>30 % normal morphology = routine criteria
METHODS FOR ANTISPERM
ANTIBODIES
SPERM VIABILITY TEST/ BLOOM’S
Mixed Agglutination Reaction (MAR)
TEST/ EOSIN-NIGROSINE TEST
 Screening procedure used primarily to
 Evaluated by mixing the specimen with
detect the presence of IgG antibodies
an eosin- nigrosine stain, then count the
number of dead cells in 100 sperm using a PROCEDURE:
brightfield or phase- contrast microscope
 Semen sample containing motile sperm is
LIVING CELLS - remains blueish- white incubated with IgG AHG.
color
 Add a suspension of latex particles or
DEAD CELLS – stains red against purple treated RBCs coated IgG
background
 Check for microscopic clumps of sperm
CHEMICAL EXEMPTION and particles
 Seminal Fluid Fructose Immunobead test
 Assesses the function of the seminal  A more specific procedure in that it can
vesicles and is used when the sperm be used to detect presence of IgG, IgM and
concentration is low IgA antibodies and demonstrate what area of
the sperm and the autoantibodies are
 Low specimen concentration may be due
affecting
to low or absent fructose level in semen
 The sperm are mixed with polyacrylamide
Resorcinol test - produces orange color
beads known to be coated with either anti-
when fructose is present
IgG, anti- IgM or anti- IgA
Using the spectrophotometric methods
 POSITIVE RESULT - shows attachment
normal fructose level is >13 umol/ ejaculate
of beads to sperm at particular areas
NOTE: specimens for fructose levels should
Microbial Testing
be tested within 2 hours or frozen to prevent
fructolysis.  Performed only if there is infection within
the reproductive system
 Anti- sperm Antibodies
Most frequently performed:
 Can be present in both men and women
 Routine aerobic and anaerobic cultures
 The presence of antibodies in the male
and test for Chlamydia trachomatis,
partner can be suspected when clumps of
SPERM FUNCTION TEST
sperms are observed during routine
TEST DESCRIPTION
urinalysis.
HAMSTER EGG Sperm are incubated with
PENETRATION speciesnonspecific hamster eggs
and penetration is observed
Mycoplasmahmoninis, and Ureaplasma evaluated for membrane integrity
urealyticum and sperm viability
OTHER CHEMICAL TEST: IN VITRO Evaluation of the acrosome to
ACROSOME produce enzymes essential for
Decreased level of neutar a- glucosidase, REACTION ovum penetration
L-carnitine and Glycerophocholine
 Suggest disorder of the epididymis
Reference Semen Chemical Values
 Decreased level of citrate, glutamyl
Neutral α glucosidase ≥ 20 mU/ejaculate
transpeptidase, zinc and acidic phosphatase
 Indicate a lack of prostatic fluid Zinc ≥ 2.4 µmol/ejaculate
Florence test Citric acid ≥ 52 µmol/ejaculate
Acid phosphatase ≥ 200 Units/ejaculate
 Test for choline
 Reagents: potassium iodide and iodine
crystal
POSITIVE RESULT: brown rhombic
crystals
Barbiero’s test MEDICO- LEGAL PURPOSE OF SEMEN

 Test for spermine  Semen analysis is done in cases of alleged

rape
 Reagents; picric acid and TCA (trichloric
acetic acid) Microscpic sperm determination

POSITIVE RESULT: yellow lift like  Enhance with xylene and examine under
structures phase-contrast microscope
 Detect presence of ACP

 Spinbarkeit test  Detection of seminal glycoprotein p30

 Test for the tenacity of mucus POST- VASECTOMY SEMEN

ANALYSIS
 Sims Huhner test
 Much less involved procedure when
 Test for the ability of sperm cells to compared to infertility testing
penetrate the cervical organ
 Concerned more of the presence or
microscopically absence of spermatozoa
CERVICAL Observation of sperm’s ability to **Specimens are routinely tested at monthly
MUCUS penetrate partner’s midcycle interval, beginning at 2 months post
PENETRATION cervical mucus vasectomy and containing 2 consecutive
HYPO-OSMOTIC Sperm exposed to monthly specimens show no spermatozoa.
SWELLING lowsodium concentrations are